Characterization of female gametophyte defects in the mutant

Megagametogenesis involves the establishment of the functional megaspore, three rounds of mitosis, establishment of female gametophyte polarity, central vacuole formation, nuclear migration, cellularization, cell specification and differentiation, nuclear fusion of the two polar nuclei and cell death of the antipodal cells and of the synergid cells. Mutations in the genes implicated in any of these processes can yield a female gametophyte mutant. In addition, mutants that have no visible defects in the embryo sacs, but that exhibit problems in pollen tube guidance, fertilization, or early embryo development can be observed. In order to study the nature of the female defect, whole mount ovules are initially observed by Nomarski/DIC microscopy after clearing, allowing relatively rapid observations of large numbers of ovules at different stages of development. Preliminary observations showed that mutants with reduced female transmission were of three general types: (1) Defective in megaspore specification or function; (2) Defective in embryo sac development and (3) Embryo sac development appears normal, but defective in subsequent fertilization and/or embryogenesis.

Phenotype of mutants observed by DIC microscopy . A, Embryo sac development is arrested at the 2 nuclear stage. Arrowheads indicate the nuclei. B , Embryo sac development is arrested at the 4 nuclear stage. C, Mature wild-type embryo sac. D , Polar nuclei fail to fuse (polar nuclei are indicated with arrows). E , embryo development arrested at 1 cell zygotic stage (arrow). Endosperm development is also affected (endosperm nuclei are indicated by arrowheads). F , Wild-type control embryo at same stage.

Gametophyte Phenotypes and Flanking Sequences

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